The human genome contains protein coding genes, highly conserved non-coding elements, enhancer elements, insulator elements, and expansive transcriptionally silenced domains. Coordinated regulation of these genomic regions determines cellular identity. Determining the identity of proteins that interact with the genome is essential or understanding how the genome is regulated. A critical barrier to this goal has been the inability to purify and identify protein complexes on DNA while preserving native interactions. The ChIP-spec (Chromatin ImmunoPurification coupled to mass spectrometry) technology has the potential to overcome this problem by offering sequence specific purification of genomic loci and identification of bound regulatory factors in their native chromatinized context. The ChIP-spec technology works by introducing a DNA sequence specific molecular handle comprised of a DNA binding TAL (Transcription-Activator Like) affinity tag (TAL-tag) into a cell type of interest. After cross linking to preserve protein-protein and protein-DNA interactions, the chromatinized genome is sheared and the TAL-tag protein purified along with proteins bound to the targeted genomic region. Known and novel proteins are then identified by either standard immunodetection techniques or quantitative mass spectrometry. We propose to develop and deploy this novel technique to purify proteins and protein complexes that are bound to genomic loci in their native chromatinized context. This ChIP-spec technique will provide a powerful approach to dissecting genome regulatory mechanisms in development and disease by isolating and identifying putative regulatory proteins. We will develop our technology using well-established human model cell lines and deploy the technique in a test case to measure dynamic exchange of proteins during cellular differentiation. To reach these goals, we propose to 1) Develop the ChIP-spec technology to defined endpoints of affinity tag targeting, site specificity, and immunopurification using human model cell lines, 2) Couple the ChIP-spec technology to quantitative proteomic technologies to enable de novo detection of genomic regulatory proteins, and 3) Deploy the ChIP-spec assay to map protein complex dynamics during cellular differentiation. Purification of transcriptional regulatory complexes in human embryonic stem cells, somatic cells, and disease model cells are likely to provide new insights into how transcriptional regulation and the cell signaling contributes to cell specification and development. These complexes will also provide the foundation for future work developing gene regulatory models and motif finding algorithms. In medicine, this technique may facilitate new therapeutic approaches and provide new therapeutic targets for human disease. Finally, this work will determine the DNA binding specificities of TAL proteins in a human genomic context. Such information will be invaluable to human gene targeting and therapeutic genome editing efforts.